Mitochondrially targeted antioxidants

ABSTRACT

The invention provides mitochondrially targeted antioxidant compounds. A compound of the invention comprises a lipophilic cation covalently coupled to an antioxidant moiety. In preferred embodiments, the lipophilic cation is the triphenyl phosphonium cation, and the compound is of the formula P(Ph 3 ) + XR.Z −  where X is a linking group, Z is an anion and R is an antioxidant moiety. Also provided are pharmaceutical compositions containing the mitochondrially targeted antioxidant compounds, and methods of therapy or prophylaxis of patients who would benefit from reduced oxidative stress, which comprise the step of administering the compounds of the invention.

TECHNICAL FIELD

[0001] The invention relates to antioxidants having a lipophiliccationic group and to uses of these antioxidants, for example, aspharmaceuticals.

BACKGROUND OF THE INVENTION

[0002] Oxidative stress contributes to a number of human degenerativediseases associated with ageing, such as Parkinson's disease, andAlzheimer's disease, as well as to Huntington's Chorea, diabetes andFriedreich's Ataxia, and to non-specific damage that accumulates withaging. It also contributes to inflammation and ischaemic-reperfusiontissue injury in stroke and heart attack, and also during organtransplantation and surgery. To prevent the damage caused by oxidativestress a number of antioxidant therapies have been developed. However,most of these are not targeted within cells and are therefore less thanoptimally effective.

[0003] Mitochondria are intracellular organelles responsible for energymetabolism. Consequently, mitochondrial defects are damaging,particularly to neural and muscle tissues which have high energydemands. They are also the major source of the free radicals andreactive oxygen species that cause oxidative stress inside most cells.Therefore, the applicants believe delivering antioxidants selectively tomitochondria will be more effective than using non-targetedantioxidants. Accordingly, it is towards the provision of antioxidantswhich may be targeted to mitochondria that the present invention isdirected.

[0004] Lipophilic cations may be accumulated in the mitochondrial matrixbecause of their positive charge (Rottenberg, (1979) Methods Enzymol,55, 547-560; Chen, (1988) Annu Rev Cell Biol 4, 155-181). Such ions areaccumulated provided they are sufficiently lipophilic to screen thepositive charge or delocalise it over a large surface area, alsoprovided that there is no active efflux pathway and the cation is notmetabolised or immediately toxic to a cell.

[0005] The focus of the invention is therefore on an approach by whichit is possible to use the ability of mitochondria to concentratespecific lipophilic cations to take up linked antioxidants so as totarget the antioxidant to the major source of free radicals and reactiveoxygen species causing the oxidative stress.

SUMMARY OF THE INVENTION

[0006] In its broadest aspect, the invention provides amitochondrially-targeted antioxidant which comprises a lipophilic cationcovalently coupled to an antioxidant moiety, wherein the antioxidantmoiety is capable of being transported through the mitochondrialmembrane and accumulated within the mitochondria of intact cells, withthe proviso that the compound is not thiobutyltriphenylphosphoniumbromide.

[0007] Preferably, the lipophilic cation is the triphenylphosphoniumcation.

[0008] Preferably, the mitochondrially-targeted antioxidant has theformula

[0009] wherein Z is an anion, X is a linking group and R is anantioxidant moiety.

[0010] Preferably, X is a C₁-C₃₀, more preferably C₁-C₂₀, carbon chain,optionally including one or more double or triple bonds, and optionallyincluding one or more substituents (such as hydroxyl, carboxylic acid oramide groups) and/or unsubstituted or substituted alkyl, alkenyl oralynyl side chains.

[0011] Preferably, X is (CH₂)_(n) where n is an integer of from 1 to 20,more preferably of from about 1 to 15.

[0012] More preferably, X is an ethylene, propylene, butylene, pentyleneor decylene group.

[0013] Preferably, Z is a pharmaceutically acceptable anion.

[0014] In one particularly preferred embodiment, themitochondrially-targeted anti-oxidant of the invention has the formula

[0015] including all stereoisomers thereof.

[0016] Preferably, Z is Br. The above compound is referred to herein as“compound 1”.

[0017] In another preferred embodiment, the mitochondrially-targetedantioxidant has the general formula:

[0018] wherein:

[0019] Z is a pharmaceutically acceptable anion, preferably a halogen,

[0020] m is an integer from 0 to 3,

[0021] each Y is independently selected from groups, chains andaliphatic and aromatic rings having electron donating and acceptingproperties,

[0022] (C)_(n) represents a carbon chain optionally including one ormore double or triple bonds, and optionally including one or moresubstituents and/or unsubstituted or substituted alkyl, alkenyl oralknyl side chains, and

[0023] n is an integer of from 1 to 20.

[0024] Preferably, each Y is independently selected from the groupconsisting of alkoxy, thioalkyl, allyl, haloalkyl, halo, amino, nitro,optionally substituted aryl, or, when m is 2 or 3, two Y groups,together with the carbon atoms to which they are attached, form analiphatic or aromatic carbocyclic or heterocyclic ring fused to the arylring. More preferably, each Y is independently selected from methoxy andmethyl.

[0025] Preferably, (C)_(n) is an alkyl chain of the formula (CH₂)_(n).

[0026] In a particularly preferred embodiment, themitochondrially-targeted antioxidant of the invention has the formula

[0027] Preferably, Z is Br. The above compound is referred to herein as“mitoquinol”. The oxidised form of the compound is referred to as“mitoquinone”.

[0028] In a further aspect, the present invention provides apharmaceutical composition suitable for treatment of a patient; whowould benefit from reduced oxidative stress which comprises an effectiveamount of a mitochondrially-targeted antioxidant of the presentinvention in combination with one or more pharmaceutically acceptablecarriers or diluents.

[0029] In a further aspect, the invention provides a method of reducingoxidative stress in a cell which comprises the step of administering tosaid cell a mitochondrially targeted antioxidant as defined above.

[0030] In still a further aspect, the invention provides a method oftherapy or prophylaxis of a patient who would benefit from reducedoxidative stress which comprises the step of administering to saidpatient a mitochondrially-targeted antioxidant as defined above.

[0031] Although broadly as defined above, the invention is not limitedthereto but also consists of embodiments of which the followingdescription provides examples.

DESCRIPTION OF DRAWINGS

[0032] In particular, a better understanding of the invention will begained with reference to the accompanying drawings, in which:

[0033]FIG. 1 is a graph which shows the uptake by isolated mitochondriaof compound 1, a mitochondrially-targeted antioxidant according to thepresent invention;

[0034]FIG. 2 is a graph which shows the accumulation of compound 1 byisolated mitochondria;

[0035]FIG. 3 is a graph which shows a comparison of a compound 1 uptakewith that of the triphenylphosphonium cation (TPMP);

[0036]FIG. 4 is a graph which shows that compound 1 protectsmitochondria against oxidative damage;

[0037]FIG. 5 is a graph which compares compound 1 with vitamin E and theeffect of uncoupler and other lipophilic cations;

[0038]FIG. 6 is a graph which shows that compound 1 protectsmitochondrial function from oxidative damage;

[0039]FIG. 7 is a graph which shows the effect of compound 1 onmitochondrial function;

[0040]FIG. 8 is a graph which shows the uptake of compound 1 by cells;

[0041]FIG. 9 is a graph which shows the energisation-sensitive uptake ofcompound 1 by cells; and

[0042]FIG. 10 is a graph which shows the effect of compound 1 on cellviability.

[0043]FIG. 11 shows the UV-absorption spectrum of[10-(6′-ubiquinonyl)decyltriphenyl-phosphonium bromide] (herein referredto as “mitoquinone”) and of the reduced form of the compound[10-(6′-ubiquinolyl)decyltriphenylphosphonium bromide] (herein referredto as “mitoquinol”).

[0044]FIGS. 12A to 12D show reactions of[10-(6′-ubiquinonyl)decyltriphenylphosphonium bromide] (“mitoquinone”)and the reduced form of the compound (“mitoquinol”) with mitochondrialmembranes. Beef heart mitochondrial membranes (20 μg/ml) were suspendedin 50 mM sodium phosphate, pH 7.2 at 20° C. In panel A rotenone andantimycin were present and for the t=0 scan, then succinate (5 mM) wasadded and scans repeated at 5 minute intervals as indicated. In panel BA₂₇₅ was monitored in the presence of rotenone and antimycin and thenmitoquinone (50 μM) was added, followed by succinate (5 mM) and malonate(20 mM) where indicated. In Panel C rotenone, ferricytochrome c (50 μM)and malonate (20 mM) were present, A₂₇₅ was monitored and mitoquinol (50μM) and myxathiazol (10 μM) were added where indicated. In panel D A₅₅₀was monitored and the experiment in Panel C was repeated in the presenceof KCN. Addition of myxathiazol inhibited this rate by about 60-70%.There was no reaction between mitoquinone and succinate or NADH in theabsence of mitochondrial membranes, however mixing 50 μM mitoquinone,but not mitoquinol, with 50 μM ferricytochrome c led to some reductionof A₅₅₀;

[0045]FIG. 13 shows reactions of mitoquinol and mitoquinone withpentane-extracted mitochondrial membranes. Pentane extracted beef heartmitochondria (100 μg protein/ml) were suspended in 50 mM sodiumphosphate, pH 7.2 at 20° C. In Panel A NADH (125 μM) was added and A₃₄₀was monitored and ubiquinone-1 (UQ-1; 50 μM) added where indicated. Thiswas repeated in Panel b, except that mitoubiquinone (50 μM) was added.In Panel C pentane extracted mitochondria were incubated withmitoquinone (50 μM), A₂₇₅ was monitored and succinate (5 mM) andmalonate (20 mM) added where indicated. In Panel D pentane-extractedmitochondria were incubated with NADH (125 μM), ferricytochrome c (50μM) and A₅₅₀ was monitored and mitoquinone (50 μM) was added whereindicated. Addition of myxathiazol inhibited the rate of reduction byabout 60-70%;

[0046]FIG. 14 shows reduction of mitoquinone by intact mitochondria. Ratliver mitochondria (100 μg/ml) were incubated in 120 mM KCl, 10 mMHEPES, 1 mM EGTA, pH 7.2 at 20° C. and A₂₇₅ monitored. In panel Arotenone and succinate (5 mM) were present and mitoquinone (50 μM) wasadded where indicated. This experiment was repeated in the presence ofmalonate (20 mM) or FCCP (333 nM). In panel B glutamate and malate (5 mMof each) were present from the start and and mitoquinone (50 MM) wasadded where indicated. This experiment was repeated in the presence ofFCCP or with rotenone and FCCP. Addition of TPMP (50 μM) instead ofmitoquinone did not lead to changes in A₂₇₅;

[0047]FIG. 15 shows uptake of radiolabelled mitoquinol by energised ratliver mitochondria and its release on addition of the uncoupler FCCP;and

[0048]FIG. 16 shows the effect of mitoquinol on isolated rat livermitochondria. In A rat liver mitochondria energised with succinate wereincubated with various concentrations of mitoquinol and the membranepotential determined as a percentage of control incubations. In B therespiration rate of succinate energised mitochondria under state 4(black), state 3 (white) and uncoupled (stippled) conditions, as apercentage of control incubations.

DESCRIPTION OF THE INVENTION

[0049] As stated above, the focus of this invention is on themitochondrial targeting of compounds, primarily for the purpose oftherapy and/or prophylaxis to reduce oxidative stress.

[0050] Mitochondria have a substantial membrane potential of up to 180mV across their inner membrane (negative inside). Because of thispotential, membrane permeant, lipophilic cations accumulateseveral-hundred fold within the mitochondrial matrix.

[0051] The applicants have now found that by covalently couplinglipophilic cations (preferably the lipophilic triphenylphosphoniumcation) to an antioxidant the compound can be delivered to themitochondrial matrix within intact cells. The antioxidant is thentargeted to a primary production site of free radicals and reactiveoxygen species within the cell, rather than being randomly dispersed.

[0052] In principle, any lipophilic cation and any antioxidant capableof being transported through the mitochondrial membrane and accumulatedwithin the mitochondria of intact cells, can be employed in forming thecompounds of the invention. It is however preferred that the lipophiliccation be the triphenylphosphonium cation herein exemplified, and thatthe lipophilic cation is linked to the antioxidant moiety by a carbonchain having 1 to 30 carbon atoms, preferably 1 to 20 carbon atoms.

[0053] While it is generally preferred that the carbon chain is analkylene group (preferably C₁-C₂₀, more preferably C₁-C₁₅), carbonchains which optionally include one or more double or triple bonds arealso within the scope of the invention. Also included are carbon chainswhich include one or more substituents (such as hydroxyl, carboxylicacid or amide groups), and/or include one or more side chains orbranches, selected from unsubstituted or substituted alky, alkenyl oralkyl groups.

[0054] In some particularly preferred embodiments, the linking group isan ethylene, propylene, butylene, pentylene or decylene group.

[0055] Other lipophilic cations which may covalently be coupled toantioxidants in accordance with the present invention include thetribenzyl ammonium and phosphonium cations.

[0056] Preferred antioxidant compounds of the invention, including thoseof general formulae I and II as defined above, can be readily prepared,for example, by the following reaction:

[0057] The general synthesis strategy is to heat a halogenatedprecursor, preferably a brominated or iodinated precursor (RBr or RI) inan appropriate solvent with 2-3 equivalents of triphenylphosphine underargon for several days. The phosphonium compound is then isolated as itsbromide or iodide salt. To do this the solvent is removed, the productis then triturated repeatedly with diethyl ether until an off-whitesolid remains. This is then dissolved in chloroform and precipitatedwith diethyl ether to remove the excess triphenylphosphine. This isrepeated until the solid no longer dissolves in chloroform. At thispoint the product is recrystallised several times from methylenechloride/diethyl ether.

[0058] It will also be appreciated that the anion of the antioxidantcompound thus prepared, which will be a halogen when this syntheticprocedure is used, can readily be exchanged with anotherpharmaceutically or pharmacologically acceptable anion, if this isdesirable or necessary, using ion exchange chromatography or othertechniques known in the art.

[0059] The same general procedure can be used to make a wide range ofmitochondrially targeted compounds with different antioxidant moieties Rattached to the triphenylphosphonium (or other lipophilic cationic)salt. These will include a series of vitamin E derivatives, in which thelength of the bridge linking the Vitamin-E function with thetriphenylphosphonium salt is varied. Other antioxidants which can beused as R include chain breaking antioxidants, such as butylatedhydroxyanisole, butylated hydroxytoluene, quinols (including those offormula II as defined above) and general radical scavengers such asderivatised fullerenes. In addition, spin traps, which react with freeradicals to generate stable free radicals can also be synthesized. Thesewill include derivatives of 5,5-dimethylpyrroline-N-oxide,tert-butylnitrosobenzene, tert-nitrosobenzene,α-phenyl-tert-butylnitrone and related compounds.

[0060] In some preferred embodiments of the invention, the antioxidantcompound is a quinol derivative of the formula II defined above. Aparticularly preferred quinol derivative of the invention is thecompound mitoquinol as defined above. Another preferred compound of theinvention is a compound of formula II in which (C)_(n) is (CH₂)₅, andthe quinol moiety is the same as that of mitoquinol.

[0061] Once prepared, the antioxidant compound of the invention, in anypharmaceutically appropriate form and optionally includingpharmaceutically-acceptable carriers or additives, will be administeredto the patient requiring therapy and/or prophylaxis. Once administered,the compound will target the mitochondria within the cell.

[0062] Set out below are synthetic schemes which may be used to preparesome other specific mitochondrially targeted antioxidant compounds ofthe present invention, namely (1) a mitochondrially targeted version ofbuckminsterfullerene; (2) a mitochondrially targeted spin trap compound;and (3) a further synthetic route for a mitochondrially targeted spintrap compound.

Buckminsterfullerene Synthesis

[0063]

[0064] The invention will now be described in more detail with referenceto the following non- limiting examples.

EXAMPLES Example 1

[0065] Experimental

[0066] 1. Synthesis of a mitochondrially-targeted vitamin-E derivative(Compound 1)

[0067] The synthesis strategy for a mitochondrially-targeted vitamin-Ederivative (compound 1) is as follows. The brominated precursor(compound 2)2-(2-bromoethyl)-3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyranwas synthesized by bromination of the corresponding alcohol as describedby Grisar et al, (1995) (J Med Chem 38, 2880-2886). The alcohol wassynthesized by reduction of the corresponding carboxylic acid asdescribed by Cohen et al., (1979) (J. Amer Chem Soc 101, 6710-6716). Thecarboxylic acid derivative was synthesized as described by Cohen et al.,(1982) (Syn Commun 12, 57-65) from2,6-dihydroxy-2,5,7,8-tetramethylchroman, synthesized as described byScott et al., (1974) (J. Amer. Oil Chem. Soc. 101,6710-6716).

[0068] For the synthesis of compound 1, 1 g of compound 2 was added to 8ml butanone containing 2.5 molar equivalents of triphenylphosphine andheated at 100° C. in a sealed Kimax tube under argon for 7-8 days. Thesolvent was removed under vacuum at room temperature, the yellow oiltriturated with diethyl ether until an off-white solid remained. Thiswas then dissolved in chloroform and precipitated with diethyl ether.This was repeated until the solid was insoluble in chloroform and it wasthen recrystallised several times from methylene chloride/diethyl etherand dried under vacuum to give a white hygroscopic powder.

[0069] 2. Mitochondrial uptake of compound 1

[0070] To demonstrate that this targeting is effective, the exemplaryvitamin E compound 1 was tested in relation to both isolatedmitochondria and isolated cells. To do this a [³H]-version of compound 1was synthesized using [³H]-triphenylphosphine and the mitochondrialaccumulation of compound 1 quantitated by scintillation counting(FIG. 1) (Burns et al., 1995, Arch Biochem Biophys 332,60-68; Bums andMurphy, 1997, Arch Biochem Biophys 339, 33-39). To do this rat livermitochondria were incubated under conditions known to generate amitochondrial membrane potential of about 180 mV (Bums et al., 1995;Burns and Murphy, 1997). Under these conditions compound 1 was rapidly(<10 s) taken up into mitochondria with an accumulation ratio of about6,000. This accumulation of compound 1 into mitochondria was blocked byaddition of the uncoupler FCCP (carbonylcyanide-p-trifluoromethoxyphenylhydrazone) which prevents mitochondriaestablishing a membrane potential (FIGS. 1 and 2) (Burns et al., 1995).Therefore compound 1 is rapidly and selectively accumulated intomitochondria driven by the mitochondrial membrane potential and thisaccumulation results in a concentration of the compound withinmitochondria several thousand fold higher than in the external medium.This accumulation is rapidly (<10 s) reversed by addition of theuncoupler FCCP to dissipate the mitochondrial membrane potential afteraccumulation of compound 1 within the mitochondria. Therefore themitochondrial specific accumulation is solely due to the mitochondrialmembrane potential and is not due to specific binding or covalentinteraction.

[0071] The mitochondrial specific accumulation of compound 1 also occursin intact cells. This was measured as described by Burns and Murphy,1997 and the accumulation was prevented by dissipating both themitochondrial and plasma membrane potentials. In addition, compound 1was not accumulated by cells containing defective mitochondria, whichconsequently do not have a mitochondrial membrane potential. Thereforethe accumulation of compound 1 into cells is driven by the mitochondrialmembrane potential.

[0072] The accumulation ratio was similar across a range ofconcentrations of compound 1 and the amount of compound 1 taken insidethe mitochondria corresponds to an intramitochondrial concentration of4-8 mM (FIG. 2). This uptake was entirely due to the membrane potentialand paralleled that of the simple triphenylphosphonium cation TPMP overa range of membrane potentials (FIG. 3). From comparison of the uptakeof TPMP and compound 1 at the same membrane potential we infer thatwithin mitochondria about 84% of compound 1 is membrane-bound (cf. About60% for the less hydrophobic compound TPMP).

[0073] Further details of the experimental procedures and results aregiven below.

[0074]FIG. 1 shows the uptake of 10 μM [3H] compound 1 by energised ratliver mitochondria (continuous line and filled symbols). The dotted lineand open symbols show the effect of addition of 333 nM FCCP at 3 min.Incubation with FCCP from the start of the incubation led to the sameuptake as for adding FCCP at 3 min (data not shown). Liver mitochondriawere prepared from female Wistar rats by homogenisation followed bydifferential centrifugation in medium containing 250 mM sucrose, 10 mMTris-HCL (pH 7.4) and 1 mM EGTA and the protein concentration determinedby the biuret assay using BSA as a standard. To measure [³H] compound 1uptake mitochondria (2 mg protein/ml) were suspended at 25° C. in 0.5-1ml 110 mM KCl, 40 mM Hepes-KOH, pH 7.2, 0.1 mM EDTA supplemented withnigericin (1 μg/ml), 10 mM succinate, rotenone 1.33 μg/ml and 60 nCi/ml[³H] compound 1 and 10 μM compound 1. After the incubation mitochondriawere pelleted by centrifugation and the [3H] compound 1 in thesupernatant and pellet quantitated by scintilation counting.

[0075]FIG. 2 shows the mitochondrial accumulation ratios [(compound 1/mgprotein)/(compound 1/μl)] obtained following 3 min incubation ofenergised rat liver mitochondria with different concentrations ofcompound 1 (filled bars) and the effect of 333 nM FCCP on these (openbars). The dotted line and open circles show compound 1 uptake bymitochondria, corrected for FCCP-insensitive binding. To measure [³H]compound 1 accumulation ratio mitochondria (2 mg protein/ml) weresuspended at 25° C. in 0.5-1 ml 110 mM KCl, 40 mM Hepes-KOH, pH 7.2, 0.1mM EDTA supplemented with nigericin (1 μg/ml), 10 mM succinate, rotenone1.33 μg/ml and 6-60 nCi/ml [³H] compound 1 and 1-50 μM compound 1. Afterthe incubation mitochondria were pelleted by centrifugation and the [3H]compound 1 in the supernatant and pellet quantitated by scintillationcounting.

[0076]FIG. 3 shows a comparison of compound 1 uptake with that of TPMPat a range of mitochondrial membrane potentials. Energised rat livermitochondria were incubated for 3 min with 10 μM compound 1 and 1 μMTPMP and different membrane potentials established with 0-8 mM malonateor 333 nM FCCP. The accumulation ratios of parallel incubations witheither 60 nCi/ml [³H] compound 1 or 50 nCi/ml [³H] TPMP were determined,and the accumulation ratio for compound 1 is plotted relative to that ofTPMP at the same membrane potential (slope=2.472, y intercept=319,r=0.97). Mitochondria (2 mg protein/ml) were suspended at 25° C. in0.5-1 ml 10 mM KCl, 40 mM Hepes-KOH, pH 7.2, 0.1 mM EDTA supplementedwith nigericin (1g/ml), 10 mM succinate, rotenone 1.33 μg/ml.

[0077] 3. Anti-oxidant efficacy of compound 1

[0078] The compounds of the invention are also highly effective againstoxidative stress. To demonstrate this, exemplary compound 1 was furthertested using rat brain homogenates. The rat brain homogenates wereincubated with or without various concentrations of the test compounds(compound 1; native Vitamin E (α-tocopherol), bromobutyltriphenylphosphonium bromide, Trolox (a water soluble form of Vitamin E)and compound 2,ie2-(2-bromoethyl)-3,4-dihydro-2,5,7,8-tetramethyl-2H-l-benzopyran-6-ol, the precursor of compound 1 (“Brom Vit E”)) and the oxidativedamage occurring over the incubation was quantitated using the TBARSassay (Stocks et al., 1974, Clin Sci Mol Med 47,215-222). From this theconcentration of compound required to inhibit oxidative damage by 50%was determined. In this system 210 nM compound 1 inhibited oxidativestress by 50% while the corresponding value for native Vitamin E was 36nM. The value for bromobutyltriphenylphosphonium bromide, which containsthe triphenylphosphonium moiety but not the antioxidant Vitamin E moietywas 47 μM. These data show that compound 1 is an extremely effectiveantioxidant, within an order of magnitude as effective as Vitamin E.Comparison with bromobutyltriphenylphosphonium bromide shows that theantioxidant capability is due to the Vitamin E function and not to thephosphonium salt. Further details of the experimental procedures andresults are set out below.

[0079] The IC₅₀ values for inhibition of lipid peroxidation weredetermined in rat brain homogenates, and are means±SEM or range ofdeterminations on 2-3 brain preparations. Octan-1-ol/PBS partitioncoefficients are means±SEM for three independent determinations. N.D.not determined. Partition coefficients were determined by mixing 200 μMof the compound in 2 ml water-saturated octanol-1-ol with 2 mloctanol-saturated-PBS at room temperature for 1 h, then the two layerswere separated by brief centrifugation and their concentrationsdetermined spectrophotometrically from standard curves prepared in PBSor octanol. To measure antioxidant efficacy four rat brains werehomogenised in 15 ml 40 mM potassium phosphate (pH 7.4), 140 mM NaCl at4° C., particulate matter was pelleted (1,000×g at 4° C. for 15 min) andwashed once and the combined supernatants stored frozen. Aliquots wererapidly thawed and 5 mg protein suspended in 800 μl PBS containingantioxidant or ethanol carrier and incubated at 37° C. for 30 min.Thiobarbituric acid reactive species (TBARS) were quantitated at 532 nmby adding 200 μl conc. HClO₄ and 200 μl 1% thiobarbituric acid to theincubation, heating at 100° C. for 15 min and then cooling andclarification by centrifugation (10,000×g for 2 min). The results areshown in Table 1 below. TABLE 1 Partition coefficients and antioxidantefficacy of compound 1 and related compounds IC₅₀ for inhibition oflipid Octanol:PBS partition Compound peroxidation (nM) coefficientCompound 1 210 ± 58  7.37 ± 1.56 Bromo Vit E 45 ± 26 33.1 ± 4.4 α-Tocopherol 36 ± 22 27.4 ± 1.0  Trolox 18500 ± 5900  N.D. BrBTP 47000 ±13000 3.83 ± 0.22

[0080] When mitochondria were exposed to oxidative stress compound 1protected them against oxidative damage, measured by lipid peroxidationand protein carbonyl formation (FIG. 4). This antioxidant protection wasprevented by incubating mitochondria with the uncoupler FCCP to preventuptake of compound 1, and lipophilic cations alone did not protectmitochondria (FIG. 5). Most importantly, the uptake of compound 1protected mitochondrial function, measured by the ability to generate amembrane potential, far more effectively than Vitamin E itself (FIG. 6This shows that the accumulation of compound 1 into mitochondriaselectively protects their function from oxidative damage. In addition,we showed that compound 1 is not damaging to mitochondria at theconcentrations that afford protection (FIG. 7).

[0081] The next step was to determine whether compound 1 was accumulatedby intact cells. Compound 1 was rapidly accumulated by intact 143Bcells, and the amount accumulated was greater than that by ρ° cellsderived from 143B cells. This is important because the ρ° cells lackmitochondrial DNA and consequently have far lower mitochondrial membranepotential than the 143B cells, but are identical in every other way,including plasma membrane potential, cell volume and protein content(FIG. 8); this suggests that most of the compound 1 within cells ismitochondrial. A proportion of this uptake of compound 1 into cells wasinhibited by blocking the plasma and mitochondrial membrane potentials(FIG. 9). This energisation-sensitive uptake corresponds to an intramitochondrial concentration of compound 1 of about 2-4 mM, which issufficient to protect mitochondria from oxidative damage. Theseconcentrations of compound 1 are not toxic to cells (FIG. 10).

[0082] Further details of the experimental procedures and results arediscussed below.

[0083]FIG. 4 shows the protection of mitochondria against oxidativedamage by compound 1. Mitochondria were exposed to oxidative stress byincubation with iron/ascorbate and the effect of compound 1 on oxidativedamage assessed by measuring TBARS (filled bars) and protein carbonyls(open bars). Rat liver mitochondria (10 mg protein) were incubated at25° C. in a shaking water bath in 2 ml medium containing 100 mM KCl, 10mM Tris, pH 7.7, supplemented with rotenone (1.33 μg/ml), 10 mMsuccinate, 500 μM ascorbate and other additions. After preincubation for5 min, 100 μM FeSO₄ was added and 45-55 min later duplicate samples wereremoved and assayed for TBARS or protein carbonyls.

[0084]FIG. 5 shows a comparison of compound 1 with vitamin E and theeffect of uncoupler and other lipophilic cations. Energised rat livermitochondria were exposed to tert-butylhydroperoxide and the effect ofcompound 1 (filled bars), o-tocopherol (open bars), compound 1+333 nMFCCP (stippled bars) or the simple lipophilic cation bromobutyltriphenylphosphonium (cross hatched bars) on TBARS formation determined.Rat liver mitochondria (4 mg protein) were incubated in 2 ml mediumcontaining 120 mM KCl, 10 mM Hepes-HCl pH 7.2; 1 mM EGTA at 37° C. in ashaking water bath for 5 min with various additions, then tert butylhydroperoxide (5 mM) was added, and the mitochondria incubated for afurther 45 min and then TBARS determined.

[0085]FIG. 6 shows how compound 1 protects mitochondrial function fromoxidative damage. Energised rat liver mitochondria were incubated withiron/ascorbate with no additions (stippled bars), 5 μM compound 1(filled bars), 5 μM α-tocopherol (open bars) or 5 μM TPMP (cross hatchedbars), and then isolated and the membrane potential generated byrespiratory substrates measured relative to control incubations in theabsence of iron/ascorbate. Rat liver mitochondria were incubated at 25°C. in a shaking water bath in 2 ml medium containing 100 mM KCl, 10 mMTris, pH 7.7, supplemented with rotenone (1.33 μg/ml), 10 mM succinate,500 μM ascorbate and other additions. After preincubation for 5 min, 100μM FeSO₄ was added and after 30 min the incubation was diluted with 6 mlice-cold STE 250 mM sucrose, 10 mM Tris-HCL (pH 7.4) and 1 mM EGTA,.pelleted by centrifugation (5 min at 5,000×g) and the pellet resuspendedin 200 μl STE and 20 μl (=1 mg protein) suspended in 1 ml 110 mM KCl, 40mM HEPES, 0.1 M EDTA pH 7.2 containing 1 μM TPMP and 50 nCi/ml [3H] TPMPeither 10 mM glutamate and malate, 10 mM succinate and rotenone, or 5 mMascorbate/ 100 μM TMPD with rotenone and myxothiazol (2 μg/ml),incubated at 25° C. for 3 min then pelleted and the membrane potentialdetermined as above and compared with an incubation that had not beenexposed to oxidative stress.

[0086]FIG. 7 shows the effect of compound 1 on the membrane potential(filled bars) and respiration rate of coupled (open bars),phosphorylating (stippled bars) and uncoupled mitochondria (crosshatched bars), as a percentage of values in the absence of compound 1.The effect of various concentrations of compound 1 on the membranepotential of isolated mitochondria was determined from the distributionof [³H] TPMP by incubating rat liver mitochondria (2 mg protein/ml) in0.5 ml medium as above containing 1 μM TPMP and 50 nCi/ml [³H] TPMP at25° C. for 3 min. After the incubation mitochondria were pelleted bycentrifugation and the [³H] TPMP in the supernatant and pelletquantitated by scintilation counting and the membrane potentialcalculated assuming a volume of 0.5 μl/mg proteins and that 60% ofintramitochondrial TPMP is membrane bound. To measure the effect ofcompound 1 on coupled, phosphorylating and uncoupled respiration rates,mitochondria (2 mg protein/ml) were suspended in 120 mM KCl, 10 mMHepes-HCl pH 7.2, 1 mM EGTA, 10 mM K Pi in a 3 ml Clark oxygen electrodethen respiratory substrate, ADP (200 μM) and FCCP (333 nM) were addedsequentially to the electrode and respiration rates measured.

[0087]FIG. 8 shows the uptake of compound 1 by cells. Here 10⁶ 143Bcells (closed symbols) or ρ° cells (open symbols) were incubated with 1μM [3H] compound 1 and the compound 1 accumulation ratio determined.Human osteosarcoma 143B cells and a derived ρ° cell line lackingmitochondrial DNA were cultured in DMEM/10% FCS (foetal calf serum)supplemented with uridine and pyruvate under an atmosphere of 5% CO₂/95%air at 37° C., grown to confluence and harvested for experiments bytreatment with trypsin. To measure [³H] compound 1 accumulation cells(10⁶) were incubated in 1 ml HEPES-buffered DMEM. At the end of theincubation, cells were pelleted by centrifugation, the cell pellet andthe supernatant prepared for scintillation counting and the accumulationratio [compound I/mg protein)/(compound 1/μl)] calculated.

[0088]FIG. 9 shows the amount of compound 1 taken up by 10⁶ 143B cellsover 1 h incubation, corrected for inhibitor-insensitive binding. Humanosteosarcoma 143B cells were incubated in 1 ml HEPES-buffered DMEM with1-50 μM compound 1 supplemented with 6-60 nCi/ml [³H] compound 1. Todetermine the energistration-dependent uptake, parallel incubations with12.5 μM oligomycin, 20 μM FCCP, 10 μM myxathiazol, 100 nM valinomycinand 1 mM ouabain were carried out. At the end of the incubation, cellswere pelleted by centrifugation and prepared for scintillation countingand the energisation-sensitive uptake determined.

[0089]FIG. 10 shows the effect of compound 1 on cell viability. Here,confluent 143B cells in 24 well tissue culture dishes were incubatedwith various concentrations of compound 1 for 24 h and cell viabilitymeasured by lactate dehydrogenase release.

Example 2 Synthesis of [10-(6′-ubiquinolyl)decyltriphenylphosphoniumbromide] (herein referred to as “mitoquinol”)

[0090] Synthesis of precursors

[0091] To synthesise 11-bromoundecanoic peroxide 11-bromoundecanoic acid(4.00 g, 15.1 mmol) and SOCl₂ (1.6 mL, 21.5 mmol) were heated, withstirring, at 90° C. for 15 min. Excess SOCl₂ was removed by distillationunder reduced pressure (15 mm Hg, 90° C.) and the residue (IR, 1799cm⁻¹) was dissolved in diethyl ether (20 mL) and the solution cooled to0° C. Hydrogen peroxide (30%/1.8 mL) was added, followed by dropwiseaddition of pyridine (1.4 mL) over 45 min. Diethyl ether (10 mL) wasadded and the mixture was stirred for 1 h at room temperature thendiluted with diethyl ether (150 mL) and washed with H₂O (2×70 mL), 1.2 MHCl (2×70 mL), H₂O (70 mL), 0.5 M NaHCO₃ (2×70 mL) and H₂O (70 mL). Theorganic phase was dried over MgSO₄ and the solvent removed at roomtemperature under reduced pressure, giving a white solid (3.51 g). IR(nujol mull) 1810, 1782.

[0092] 6-(10-bromodecyl)ubiquinone was synthesised by mixing crudematerial above (3.51 g, 12.5 mmol max), (ubiquinoneo, 1.31 g, 7.19 mmol,Aldrich) and acetic acid (60 mL) and stirring the mixture for 20 h at100° C. The mixture was diluted with diethyl ether (600 mL) and washedwith H₂O (2×400 mL), 1M HCl (2×450 mL), 0.50 M NaHCO₃ (2×450 mL) and H₂O(2×400 mL). The organic phase was dried over MgSO₄. The solvent wasremoved under reduced pressure, giving a reddish solid (4.31 g). Columnchromatography of the crude solid on silica gel (packed in CH₂Cl₂) andelution with CH₂Cl₂ gave the product as a red oil (809 mg, 28%) andunreacted ubiquinone as a red solid (300 mg, 1.6 mmol, 13%). TLC: R_(f)(CH₂Cl₂, diethyl ether 20:1) 0.46; IR (neat) 2928, 2854, 1650, 1611,1456, 1288;λ_(max) (ethanol): 278 nm; 1H NMR (299.9 MHz) 3.99 (s, 6H,2×—OCH₃), 3.41 (t, J=6.8 Hz, 3H, -CH₂-Br), 2.45 (t, J=7.7 Hz, 2H,ubquinone -CH₂—), 2.02, (s, 3H, —CH₃). 1.89 (quin, J=7.4 Hz, 3H,—CH₂—CH₂—Br), 1.42-1.28 (m, 20H, —(CH₂)₇—); ¹³C NMR (125.7 MHz) 184.7(carbonyl), 184.2 (carbonyl), 144.3 (2C, ring), 143.1 (ring), 138.7(ring), 61.2 (2×-OCH₃), 34.0 (—CH₂—), 32.8 (—CH₂—), 29.8 (—CH₂—), 29.4(2×—CH₂—), 29.3 (—CH₂—), 28.7 (2×—CH₂—), 28.2 (—CH₂—), 26.4 (—CH₂—),11.9 (—CH₃). Anal. Calcd. For Cl₉H₂₉O₂Br:C, 56.86; H, 7.28; Found: C,56.49, H, 7.34; LREI mass spectrum: calcd. For Cl₉H₂₉O₂Br 400/402; Found400/402.

[0093] To form the quinol, 6-(10-bromodecyl)-ubiquinol, NaBI₄ (295 mg,7.80 mmol) was added to a solution of the quinone (649 mg, 1.62 mmol) inmethanol (6 mL) and stirred under argon for 10 min. Excess NaBH₄ wasquenched with 5% HCl (2 mL) and the mixture diluted with diethyl ether(40 mL). The organic phase was washed with 1.2 M HCl (40 mL) andsaturated NaCl (2×40 mL), and dried over MgSO₄. The solvent was removedunder reduced pressure, giving a yellow oily solid (541 mg, 83%). ¹H NMR(299.9 MHz) 5.31 (s, 1H, —OH), 5.26 (s, 1H, —OH), 3.89 (s, 6H, 2×—OCH₃),3.41 (t, J=6.8 Hz, 2H, —CH₂—Br), 2.59 (t, J=7.7 Hz, 2H ubquinol-CH₂—),2.15 (s, 3H, CH₃) 1.85 (quin, J=7.4 Hz, 2H, —CH₂—CH₂—Br), 1.44-1.21 (m,19H, —CH₂)₇—).

[0094] Synthesis of 10-(6′-ubiquinolyl)decyltriphenylphosphonium bromide(“mitoquinol”)

[0095] To synthesise 10-(6-ubiquinolyl)decyltriphenylphosphoniumbromide. To a 15 mL Kimax tube were added 6-(10-bromodecyl)ubiquinol(541 mg, 1.34 mmol), PPH₃ (387 mg, 1.48 mmol), ethanol (95%, 2.5 mL) anda stirring bar. The tube was purged with argon, sealed and the mixturestirred in the dark for 88 h at 85° C. The solvent was removed underreduced pressure, giving an oily orange residue. The residue wasdissolved in CH₂Cl₂ (2 mL) followed by addition of pentane (20 mL). Theresultant suspension was refluxed for 5 min at 50° C. and thesupernatant decanted. The residue was dissolved in CH₂Cl₂ (2 mL)followed by addition of diethyl either (20 mL). The resultant suspensionwas refluxed for 5 min at 40° C. and the supernatant decanted. TheCH₂Cl₂/diethyl ether reflux was repeated twice more. Residual solventwas removed under reduced pressure, giving crude product as a creamsolid (507 mg). ¹H NMR (299.9 MHz) 7.9-7.6 (m, 20H, -P+Ph₃), 3.89 (s,6H, 2×—OCH₃), 3.91-3.77 (m, 2H, —CH₂-P⁺Ph₃), 2.57 (t, J=7.8 Hz, 2Hubquinol-CH2—), 2.14 (s, 3H, CH₃), 1.6-1.2 (m, 23H, —(CH₂)₈—). ³¹PNMR(121.4 MHz) 25.1.

[0096] The crude product (200 mg) was oxidized to10-(6′-ubiquinonyl)decyltriphenylphosphonium bromide (the oxidised form)by stirring in CDCl₃ under an oxygen atmosphere for 13 days. Theoxidation was monitored by ¹H NMR and was complete after 13 days. Thesolvent was removed under reduced pressure and the resultant residuedissolved in CH₂Cl₂ (5 mL). Excess diethyl ether (15 mL) was added andthe resultant suspension stirred for 5 min. The supernatant was decantedand the CH₂Cl₂/diethyl ether precipitation repeated twice more. Residualsolvent was removed under reduced pressure, giving crude product as abrown sticky solid (173 mg).

[0097] The quinone was reduced to the quinol by taking a mixture ofcrude quinone and quinol (73 mg, ca. 3:1 by 1H NMR) in methanol (1 mL)was added NaBH₄ (21 mg, 0.55 mmol). The mixture was stirred slowly underan argon atmosphere for 10 min. Excess NaBH₄ was quenched with 5% HBr(0.2 mL) and the mixture extracted with CH₂Cl₂. The organic extract waswashed with H₂O (3×5 mL). Solvent was removed under reduced pressure,giving a mixture of quinone and quinol (ca 1:5 by ¹H NMR) as a paleyellow solid (55 mg).For routine preparation of the quinol form theethanolic solution, dissolve in 5 vols of water, (=1 ml) add a pinch ofNaBH4 leave on ice in the dark for 5 min, then extract 3×0.5 mldichloromethane, Wash with water/HCl etc blow off in nitrogen, dissolvein same vol of etoh and take spectrum and store at −80 under argon.Yield about 70-80%. Oxidises rapidly in air so should be prepared fresh.vortex with 1 ml 2M NaCl. Collect the upper organic phase and evaporateto dryness under a stream of N₂ and dissolve in 1 ml ethanol acidifiedto pH 2.

[0098] Synthesis of ³H-10-(6′-ubicuinonyl)decyltliphenylphosphoniumbromide

[0099] To a Kimax tube was added 6-(10-bromodecyl)ubiquinol (6.3 mg;15.6 ,mol) triphenylphosphine (4.09 mg; 15.6 umol) and 100 μl ethanolcontaining [³H] triphenylphosphine (74 gCi custom synthesis by MoravekBiochemicals, Brea, Calif., USA, Spec Ac 1 Ci/mmol) and 150 μl ethanoladded. The mixture was stirred in the dark under argon for 55h at 80° C.Then it was cooled and precipitated by adition of 5 ml diethyl ether.The orange solid was dissolved in few drops of dichloromethane and thenprecipitated with diethyl ether and the solid was washed (×4) with ˜2 mldiethyl ether. Then dissolved in ethanol to give a stock solution of 404μM which was stored at −20° C. The UV absorption spectrum and TLC wereidentical to those of the unlabelled10-(6′-ubiquinonyl)decyltriphenylphosphonium bromide and the specificactivity of the stock solution was 2.6 mCi/mmol.

[0100] Extinction coefficients

[0101] Stock solutions of the quinone in ethanol were stored at −80° C.in the dark and their concentrations confirmed by 31P nmr. The compoundwas converted to the fully oxidised form by incubation in basic 95%ethanol over an hour on ice or by incubation with beef heartmitochondrial membrane at room temperature, either procedure leading tothe same extinction coefficient of 10,400 M⁻¹ cm⁻¹ at the local maximumof 275 nm, with shoulders at 263 and 268 nm corresponding to theabsorption maxima of the triphenylphosphonium moiety (Smith et al, Eur.J. Biochem., 263, 709-716, 1999; Burns et al, Archives of Biochemistryand Biophysics, 322, 60-68, 1995) and a broad shoulder at 290 nm due tothe quinol (Crane et al, Meth Enzymol., 18C, 137-165, 1971). Reductionby addition of NaBH4 gave the spectrum of the quinol which had theexpected peak at 290 nm with an extinction coefficient of 1800 m⁻¹ cm⁻¹and the extinction coefficient for at 268 nm was 3,000 m⁻¹ cm⁻¹ the sameas that for the phosphonium moiety alone (Burns, 1995 above). Theextinction coefficient of 10,400 m⁻¹ cm⁻¹ at 275 nm was lower than thatfor other quinones which have values of 14,600 m⁻¹ cm⁻¹ in ethanol(Crane, 1971 above) and 12,250 M⁻¹ cm⁻¹ in aqueous buffer (Cabrini etal, Arch Biochem Biophys, 208, 11-19, 1981). While the absorbance of thequninone was about 10% lower in buffer than in ethanol, the discrepancywas not due to an interaction between the phosphonium and the quinone asthe absorbance of the precursor quinone before linking to thephosphonium and that of the simple phosphoniummethyltriphenylphosphonium were additive when 50 μM of each were mixedtogether in either ethanol or aqueous buffer. The Δε_(OX-red) was 7,000M⁻¹cm⁻¹ .

[0102] The spectrum of fully oxidised mitoquinone (50 iM) in 50 mMsodium phosphate, pH 7.2 is shown in FIG. 11. Addition of NaBH₄ gave thefully reduced compound, mitoquinol. The UV absorption spectrum of thereduced (quinol) and oxidised (quinone) mitoquinone/ol are shown in FIG.11. To determine whether the mitochondrial respiratory chain could alsooxidise or reduce the compound mitoquinone was incubated with beef heartmitochondrial membranes (FIG. 12). In panel A the spectrum of fullyoxidised mitoquinone in the presence of antimycin inhibited membranes isshown (t=0; FIG. 12A). Addition of succinate led to the gradualreduction of the mitoquinol as measured by repeating the measurementevery five minutes and showing that the peak at 275 nm graduallydisappeared, the presence of antimycin prevented the oxidation of thequinol by mitochondrial complex III. Succinate did not lead to thecomplete reduction of mitoquinone to mitoquinol, as can be seen bycomparing the complete reduction brought about by borohydride (FIG. 11),instead it reduced about 23% of the added ubiquinone (FIG. 12A). This ispresumably due to equilibration of the quinol/quinone couple with thesuccinate/fumarate couple (Em Q=40 mV at pH 7, Em Suc=30 mV), hence thisproportion corresponds to an Eh of about +8 mV.

[0103] The reduction of mitoquinone can be followed continuously at A₂₇₅nm (FIG. 12B). On addition to rotenone inhibited mitochondrial membranesthe small amount of mitoquinol remaining was oxidised leading to aslight increase in A₂₇₅, but on addition of the Complex II substratesuccinate mitoquinone was rapidly reduced and this reduction was blockedby malonate, an inhibitor of Complex II (FIG. 12B). The rate ofreduction of mitoquinone was 51±9.9 nmol /min/mg protein, which compareswith the rate of reduction of cytochrome c by succinate in the presenceof KCN of 359 nmol/min/mg. Allowing for the 2 electrons required formitoquinone reduction compared with 1 for cytochrome c the rate ofelectron flux into the mitoquinone pool is of similar order to theelectron flux through the respiratory chain.

[0104] To determine whether mitoquinol was oxidised by Complex III ofthe respiratory chain, mitoquinol was added to beef heart membraneswhich had been inhibited with rotenone and malonate (FIG. 12C). Themitoquinol was oxidised rapidly by membranes at an initial rate of about89±9 nmol mitoquinol/min/mg protein (mean of 2+/−range) and thisoxidation was blocked by myxathiazol an inhibitor of complex III (FIG.12C). To confirm that these electrons were being passed on to cytochromec, mitoquinol was then added to membrane supplemented withferricytochrome c and the rate of reduction of cytochrome c monitored(FIG. 12D). Addition of mitoquinol led to reduction of cytochrome c atan initial rate of about 93+/−13 nmol/min/mg (mean±/- range). This ratewas largely blocked by myxathiazol, although a small amount ofcytochrome c reduction (about 30-40%) was not blocked by myxathiazol.

[0105] Mitoquinone/ol may be picking up and donating electrons directlyfrom the active sites of the respiratory complexes, or it could beequilibrating with the endogenous mitochondrial ubiquinone pool. Toaddress this question the endogenous ubiquinone pool was removed frombeef heart mitochondria by pentane extraction. In the absence ofendogenous ubiquinone as an electron acceptor the pentane extracted beefheart mitochondria could not oxidise added NADH, but addition ofubiquinone-1, a ubiquinone analogue that can pick up electrons from theactive site of complex I, the oxidation of NADH is partially restored(FIG. 13A). Similarly, addition of mitoquinone also restored NADHoxidation indicating that mitoquinone can pick up electrons from thecomplex I active site (FIG. 13B). Succinate could also donate electronsto mitoquinone in pentane extracted beef heart mitochondrial in amalonate sensitive manner, suggesting that mitoquinone could also pickup electrons from the active site of Complex II (FIG. 13C). Finally, theeffect of the quinone on the flux of electrons to cytochrome c wasdetemined and it was shown that there was no NADH -ferricytochrome cactivity until mitoquinone was added (FIG. 13D), and this was partiallyinhibited by myxathizol (60-70%).

[0106] The next step was to see if mitoquinone also accepted electronswithin intact mitochondria (FIG. 14). When mitoquinone was added tointact energised mitochondria it was rapidly reduced (FIG. 14A). In thepresence of the uncoupler FCCP to dissipate the membrane potential therate was decreased about 2-3 fold, presumably due to the prevention ofthe uptake of the compound in to the mitochondria (FIG. 14A). Thecomplex II inhibitor malonate also decreased the rate of reduction ofmitoquinone (FIG. 14A). Use of the NADH-linked substratesglutamate/malate also led to the rapid reduction of mitoquinone byintact mitochondria which again was decreased by addition of theuncoupler FCCP (FIG. 14B). The Complex I inhibitor rotenone alsodecreased the rate of reduction of mitoquinone (FIG. 14B).

[0107] The next step was to see if mitoquinol was accumulated byenergised mitochondria. To do this a tritiated version of the compoundwas made, incubated with energised mitochondria and the amount taken upinto the mitochondria determined. It can be seen that the compound isaccumulated rapidly and that this accumulation is reversed by additionof the uncoupler FCCP (FIG. 15).

[0108] The next assays were to determine the toxicity of these compoundsto mitochondria and cells. To determine the toxicity to isolatedmitochondria the effect on membrane potential and respiration rate weremeasured (FIG. 16). It can be seen from FIG. 16 that 10 μM mitoquinolhad little effect on mitochondrial function and at 25 μM and above therewas some uncoupling and inhibition of respiration.

[0109] INDUSTRIAL APPLICATION

[0110] The compounds of the invention have application in selectiveantioxidant therapies for human patients to prevent mitochondrialdamage. This can be to prevent the elevated mitochondrial oxidativestress associated with particular diseases, such as Parkinson's disease,diabetes or diseases associated with mitochondrial DNA mutations. Theycould also be used in conjunction with cell transplant therapies forneurodegenerative diseases, to increase the survival rate of implantedcells.

[0111] In addition, these compounds could be used as prophylactics toprotect organs during transplantation, or ameliorate theischaemia-reperfusion injury that occurs during surgery. The compoundsof the invention could also be used to reduce cell damage followingstroke and heart attack or be given prophylactically to prematurebabies, which are susceptible to brain ischemia. The methods of theinvention have a major advantage over current antioxidant therapies -they will enable antioxidants to accumulate selectively in mitochondria,the part of the cell under greatest oxidative stress. This will greatlyincrease the efficacy of antioxidant therapies. Related lipophiliccations are being trialed as potential anticancer drugs and are known tobe relatively non-toxic to whole animals, therefore thesemitochondrially-targeted antioxidants are unlikely to have harmful sideeffects.

[0112] Those persons skilled in the art will appreciate that the abovedescription is provided by way of example only, and that differentlipophilic cation/antioxidant combinations can be employed withoutdeparting from the scope of the invention.

1. A mitochondrially-targeted antioxidant compound comprising alipophilic cation covalently coupled to an antioxidant moiety, whereinthe antioxidant moiety is capable of being transported through themitochondrial membrane and accumulated within the mitochondria of intactcells, with the proviso that the compound is notthiobutyltriphenylphosphonium bromide.
 2. A compound as claimed in claim1 wherein the lipophilic cation is the triphenylphosphonium cation.
 3. Amitochondrially-targeted antioxidant compound as claimed in claim 1,wherein said compound has the formula

wherein X is a linkig group, Z is an anion, and R is an antioxidantmoiety.
 4. A compound as claimed in claim 3, wherein X is a C₁ to C₃₀carbon chain, optionally including one or more double or triple bonds,and optionally including one or more substituents and/or unsubstitutedor substituted alkyl, alkenyl or alkyl side chains.
 5. A compound asclaimed in claim 4, wherein X is (CH₂)_(n), where n is an integer offrom 1 to
 20. 6. A compound as claimed in claim 5 wherein X is anethylene, propylene, butylene, pentylene or decylene group.
 7. Acompound as claimed in claim 3 wherein said compound has the formula

including all stereoisomers thereof
 8. A compound as claimed in claim 7wherein Z is Br.
 9. A compound as claimed in claim 3, having the formula

wherein: Z is a pharmaceutically acceptable anion, m is aninteger offrom 0to 3, each Y is independently selected from groups, chains andaliphatic and aromatic rings having electron donating and acceptingproperties, (C)_(n) represents a carbon chain optionally including oneor more double or triple bonds, and optionally including one or moresubstituents and/or unsubstituted or substituted alkyl, alkenyl oralkynyl side chains; and n is an integer of from 1 to
 20. 10. A compoundas claimed in claim 9, wherein (C)_(n) is an allyl chain of the formula(CH₂)_(n) wherein n is an integer of from 1 to
 20. 11. A compound asclaimed in claim 10, wherein each Y is independently selected from thegroup consisting of alkoxy, thioalkyl, alkyl, haloalkyl, halo, amino,nitro and optionally substituted aryl, or when m is 2 or 3, two Ygroups, together with the carbon atoms to which they are attached, forman aliphatic or aromatic carbocyclic or heterocyclic ring fused to thearyl ring.
 12. A compound as claim in claim 11 wherein each Y isindependently selected from methoxy and methyl.
 13. A compound asclaimed in claim 9 wherein said compound has the formula


14. A compound as claimed in claim 13 wherein Z is Br.
 15. Apharmaceutical composition suitable for the treatment of a patient whowould benefit from reduced oxidative stress, which comprises aneffective amount of a mitochondrially-targeted antioxidant as defined inclaim 1 in combination with one or more pharmaceutically acceptablecarriers or diluents.
 16. A pharmaceutical composition as claimed inclaim 15 wherein the mitochondrially-targeted antioxidant has theformula I

wherein X is a linking group, Z is an anion and R is an antioxidantmoiety.
 17. A pharmaceutical composition as claimed in claim 16 whereinthe mitochondrially targeted antioxidant compound is


18. A pharmaceutical composition as claimed in claim 15 wherein themitochondrially targeted antioxidant compound has the formula

wherein: Z is a pharmaceutically acceptable anion, m is an integer offrom 0 to 3, each Y is independently selected from groups, chains andaliphatic and aromatic rings having electron donating and acceptingproperties, (C)_(n), represents a carbon chain optionally including oneor more double ortriple bonds, and optionally including one or moresubstituents and/or unsubstituted or substituted all, alkenyl or alkynylside chains; and n is an integer of from 1 to
 20. 19. A pharmaceuticalcomposition as claimed in claim 18, wherein (C)r, is an all chain of theformula (CH₂) wherein n is an integer of from 1 to
 20. 20. Apharmaceutical composition as claimed in claim 18, wherein each Y isindependently selected from the group consisting of alkoxy, thioalkyl,ally, haloalkyl, halo, ammio, nitro and optionally substituted aryl, or,when m is 2 or 3, two Y groups, together with the carbon atoms to whichthey are attached, form an aliphatic or aromatic carbocyclic orheterocyclic ring fused to the aryl ring.
 21. A pharmaceuticallycomposition as claimed in claim 20, wherein each Y is independentlyselected from methoxy and methyl.
 22. A pharmaceutical composition asclaimed in claim 17 wherein the mitochondrially targeted antioxidantcompound is:


23. A method of therapy or prophylaxis of a patient who would benefitfrom reduced oxidative stress, which comprises the step of administeringto the patient a mitochondrially-targeted antioxidant as defmed inclaim
 1. 24. A method of reducing oxidative stress in a cell whichcomprises the step of administering to the cell amitochondrially-targeted antioxidant as defined in claim 1.